Methods to detect nucleic acids and to detect specific nucleic acids provide a foundation upon which the large and rapidly growing field of molecular biology is built. There is constant need for alternative methods and products. The reasons for selecting one method over another are varied, and include a desire to avoid radioactive materials, the lack of a license to use a technique, the cost or availability of reagents or equipment, the desire to minimize the time spent or the number of steps, the accuracy or sensitivity for a certain application, the ease of analysis, or the ability to automate the process.
The detection of nucleic acids or specific nucleic acids is often a portion of a process rather than an end in itself. There are many applications of the detection of nucleic acids in the art, and new applications are always being developed. The ability to detect and quantify nucleic acids is useful in detecting microorganisms, viruses and biological molecules, and thus affects many fields, including human and veterinary medicine, food processing and environmental testing. Additionally, the detection and/or quantification of specific biomolecules from biological samples (e.g. tissue, sputum, urine, blood, semen, saliva) has applications in forensic science, such as the identification and exclusion of criminal suspects and paternity testing as well as in genetics and medical diagnostics.
Some general methods to detect nucleic acids are not dependent upon a priori knowledge of the nucleic acid sequence. A luminescent nucleic acid detection method described in U.S. Pat. No. 4,735,897 is not sequence specific, indicates the presence of single-stranded RNA, such as mRNA. In the disclosed method, RNA is depolymerized using a polynucleotide phosphorylase (PNP) in the presence of phosphate or using a ribonuclease. That patent teaches that PNP stops depolymerizing when a double-stranded RNA segment is encountered, such as in single-stranded RNA with secondary structure, as is common in ribosomal RNA, transfer RNA, viral RNA, and the message portion of mRNA. PNP depolymerization of the polyadenylated tail of mRNA in the presence of inorganic phosphate forms ADP. Alternatively, depolymerization of RNA using a ribonuclease releases AMP. The released AMP is converted to ADP with myokinase, and ADP is converted into ATP by pyruvate kinase or creatine phosphokinase. Either the ATP or the byproduct from the organophosphate co-reactant (pyruvate or creatine) is detected as an indirect method of detecting mRNA. In U.S. Pat. No. 4,735,897, ATP is detected by a luminescence spectroscopic luciferase detection system. In the presence of ATP and oxygen, luciferase catalyzes the oxidation of luciferin, producing light that can then be quantified using a luminometer. Additional products of the reaction are AMP, pyrophosphate and oxyluciferin.
Hybridization methods to detect nucleic acids are dependent upon knowledge of the nucleic acid sequence. Many known nucleic acid detection techniques depend upon specific nucleic acid hybridization in which an oligonucleotide probe is hybridized or annealed to nucleic acid in the sample or on a blot, and the hybridized probes are detected.
Several hybridization methods to detect nucleic acids are discussed in the paragraphs that follow. These include PCR, Southern blots, and fluorescent hybridization with and without PCR. Several of hybridization methods are even useful for detecting specific nucleic acid sequences such as single nucleotide polymorphisms (SNPs), and distinguishing them from very similar sequences, and this is also discussed.
Polymerase chain reaction (PCR) and Southern blot-based hybridization methods for the detection of predetermined nucleic acid rely upon the use of hybridizing labeled primers or probes. Such probes have been labeled and detected using radioactivity; fluorescent spectroscopic methods using fluorescent dyes, acridinium esters and digoxygenin; and absorbance spectroscopic (often visible) methods using horseradish peroxidase, jack bean urease and alkaline phosphatase. PCR products made with unlabeled primers may be detected in other ways, such as electrophoretic gel separation followed by dye-based visualization.
There are hybridization assays to detect nucleic acid that involve fluorescence spectroscopic techniques utilizing energy transfer effects between fluorophores (FRET). U.S. Pat. No. 5,691,146 describes the use of fluorescent hybridization probes that are fluorescence-quenched unless they are hybridized to the target nucleic acid sequence. U.S. Pat. No. 5,723,591 describes fluorescent hybridization probes that are fluorescence-quenched until hybridized to the target nucleic acid sequence, or until the probe is digested. Such techniques provide information about hybridization, and are of varying degrees of usefulness for the determination of single base variances in sequences. Some fluorescence techniques involve digestion of the probe in a nucleic acid hybrid in a 5' to 3' direction to release a fluorophore providing a signal from proximity with a fluorescence quencher, thereby increasing the signal, for example, U.S. Pat. Nos. 5,691,146 and 5,876,930.
The fluorescence spectroscopic techniques for detection of nucleic acid hybrids have been applied to real-time (or kinetic) PCR and single nucleotide polymorphism (SNP) detection. Many of these systems are platform based and require specialized equipment, complicated primer design and expensive supporting materials for SNP detection. SNP detection using real-time PCR amplification relies on the ability to detect amplified segments of nucleic acid as they are made during the amplification reaction. Three basic real-time SNP detection methodologies exist: (i) increased fluorescence of double-stranded DNA-specific dye binding, (ii) decreased quenching of fluorescence during amplification (e.g. Taqman.RTM.), and (iii) increased fluorescence energy transfer during amplification (C. Wittwer et al., Biotechniques, 22:130-138 (1997)). All of these techniques are non-gel based and each strategy is briefly discussed below.
A variety of dyes are known to exhibit increased fluorescence in response to binding double stranded DNA. Production of wild type or mutation containing-PCR products are continuously monitored by the increased fluorescence of dyes such as ethidium bromide or syber green as they bind to the accumulating duplex PCR product. Note that dye binding is not selective for the sequence of the PCR product, and elevated levels of non-specific background can give rise to false signals with this technique.
Some technologies for real time detection of PCR products are based on detecting nucleic acid hybrids using fluorescence resonance energy transfer (FRET; mentioned above). These technologies either indirectly measure the amplification reaction through the use of a separate, labeled probe that hybridizes with but is not incorporated into the amplification product (U.S. Pat. Nos. 5,348,853; 5,119,801; 5,312,728; 5,962,233; 5,945,283; 5,876,930; 5,723,591; and 5,691,146) or directly detect amplification products through the use of a label directly incorporated in the amplification primer(s) (U.S. Pat. No. 5,866,336).
One such FRET-based technology for real time PCR product detection is known generally as 5' nuclease PCR assay (TaqMan.RTM. assay). In this assay the decrease in fluorescence quenching resulting from the cleavage of dually-labeled probes that hybridize downstream of amplification primers is monitored in an amplification reaction. A polymerase extends the growing nucleic acid chain from the amplification primers, and degrades hybridized dually-labeled probes from their 5'-termini using the 5' to 3' exonuclease activity of thermostable polymerases such as Taq DNA Polymerase. C. Wittwer et al., Biotechniques, 22:130-138 (1997); P. Holland et al, Proc. Natl. Acad. Sci., USA, 88:7276-7280 (1991). Although complementary to the PCR product, the fluorescently-labeled nucleic acid hybrid-detecting probes used in this assay are distinct from the PCR primers. The probes are dually-labeled with both a molecule capable of fluorescence and a molecule capable of quenching fluorescence. When the probes are intact, and hybridize to an amplification template, intramolecular quenching of energy between the two fluorophores (dual labels) of the probe leads to low, background levels of fluorescent signal. When a fluorescent molecule is liberated from the proximity of the fluorescence quencher by the exonuclease activity of a DNA polymerase (e.g. Taq DNA Polymerase) during amplification, the quenching is greatly reduced leading to increased fluorescent signal. This probe is degraded by the 5'-exonuclease activity of DNA polymerase when it hybridizes downstream of polymerase on the segment of DNA template being amplified.
In the TaqMan.RTM. assay, the donor and quencher are preferably located on the 3' and 5'-ends of the probe respectively, because the requirement that 5' to 3' hydrolysis be performed between the fluorophore and the quencher is met only when these two moieties are not too close to each other. Lyamichev et al., Science, 260:778-783, (1993). However, this can be a drawback of the assay since efficiency of energy transfer decreases with the inverse sixth power of the distance between the reporter and quencher. As a result, the background emissions from unhybridized probe can be high since the assay does not permit the quencher to be close enough to the reporter to achieve the most efficient quenching. In addition, not all of the probe hybridized to the PCR product will be hydrolyzed, some will be displaced without hydrolysis resulting in a loss of signal.
An additional form of real-time PCR product detection, termed molecular beacon assay, also capitalizes on the intramolecular quenching of a fluorescent molecule by use of a tethered quenching moiety. The molecular beacon technology utilizes hairpin-shaped, hybridization probe molecules with an internally-quenched fluorophore whose fluorescence is restored by binding to a DNA target of interest (Tyagi and Kramer, Nat. Biotechnol., 14:303-308 (1996), U.S. Pat. Nos. 5,119,801 and 5,312,728). When used in PCR reactions, the molecular beacon probe, which hybridizes to one of the strands of the PCR product emits fluorescence, while those that remain unhybridized are fluorescence-quenched. As a result, the amount of fluorescence will increase as the amount of PCR product increases. Increased binding of the molecular beacon probe to the accumulating PCR product can be used to specifically detect SNPs present in genomic DNA.
However, it is unlikely that the beacon probes will hybridize quantitatively to one strand of double-stranded PCR product, especially when the amplification product is much longer than the beacon probe. Even those probes that are hybridized could be displaced by the second DNA strand over a short period of time; as a result, this method cannot be quantitative.
U.S. Pat. No. 5,885,775 discloses a hybridization method to determine the presence of nucleic acids utilizing mass spectrometric analysis. The method disclosed therein for determining sequence information, including SNPs, involves hybridizing one or more oligonucleotide probes having a nucleotide sequence complementary to a portion of the sample polynucleotide to form a complex. The formed complex is then contacted with at least a member selected from the group consisting of nucleosides, dideoxynucleosides, polymerase, nucleases, transcriptases, ligases and restriction enzymes to alter at least of a subset of the oligonucleotide probes. Then the molecular weight of the altered probes is determined using mass spectrometry, and the sequence of the sample polynucleotide is inferred.
A method disclosed in U.S. Pat. No. 5,885,775 for SNP determination involves the use of an extension primer hybridized such that the 3'-terminus of the primer is at the putative point mutation, and after reacting with polymerase and nucleotides the identity of the added nucleotide in the extension product is determined using mass spectrometry. A multiplex format is also disclosed for this polymerase extension assay.
Another method disclosed in U.S. Pat. No. 5,885,775 for SNP determination involves the use of a ligase or a ligase extension, wherein two hybridized primers are linked at a putative point mutation site. A multiplex format is also disclosed.
Another method disclosed in U.S. Pat. No. 5,885,775 is a "combinatorial" chain termination sequencing method wherein primers are extended in the presence of some ddNTPs to produce a ladder. Alternatively, a ladder of different length polynucleotides is produced by treating the extension products with an exonuclease activity from one of the following enzymes: phosphodiesterase type I, exonuclease I, exonuclease III, exonuclease V, exonuclease VII, and DNA polymerase III. A multiplex format is also disclosed.
Enzymes having template-dependent polymerase activity for which some 3' to 5' depolymerization activity has been reported include E. coli DNA Polymerase (Deutscher and Kornberg, J. Biol. Chem., 244(11):3019-28 (1969)), T7 DNA Polymerase (Wong et al., Biochemistry, 30:526-37 (1991); Tabor and Richardson, J. Biol. Chem., 265: 8322-28 (1990)), E. coli RNA polymerase (Rozovskaya et al., Biochem. J., 224:645-50 (1994)), AMV and RLV reverse transcriptases (Srivastava and Modak, J. Biol. Chem., 255: 2000-4 (1980)), and HIV reverse transcriptase (Zinnen et al., J. Biol. Chem., 269:24195-202 (1994)). A template-dependent polymerase for which 3' to 5' exonuclease activity has been reported on a mismatched end of a DNA hybrid is phage 29 DNA polymerase (de Vega, M. et al. EMBO J., 15:1182-1192, 1996).
There is a need for alternative methods for the detection of nucleic acid hybrids. There is a demand for highly sensitive methods that are useful for determining the presence or absence of specific nucleic acid sequences, for example methods to determine viral load that are able to reliably detect as few as 10 copies of a virus present in a body, tissue, fluid, or other biological sample. There is a great demand for methods to determine the presence or absence of nucleic acid sequences that differ slightly from sequences that might otherwise be present. There is a great demand for highly specific methods to determine the presence or absence of sequences unique to a particular species in a sample. There is also a great demand for such methods that are rapid and more sensitive than the known methods, highly reproducible and automatable with a flexible format.
It would be beneficial if another method were available for detecting the presence of a sought-after, predetermined target nucleotide sequence or allelic variants. It would also be beneficial if such a method were operable using a sample size of the microgram to picogram scale. It would further be beneficial if such a detection method were capable of providing multiple analyses in a single assay (multiplex assays).
In contrast to the techniques discussed before, a process of this invention discussed hereinafter has been designed as a modular technology that can use a variety of instruments that are suited to the throughput needs of the end-user and have a flexible and open system architecture for detection analysis. Alternative analytical detection methods, such as mass spectrometry, absorbance and fluorescence spectroscopic detection methods are used in a process of this invention, providing additional assay flexibility. The disclosure that follows thus provides a method that provides alternatives to the existing nucleic acid hybrid detection systems.